Mutation and dysregulation of the cardiac ryanodine receptor (RyR2) or sarcoplasmic reticulum (SR) calcium release channel contributes directly to catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure (HF) in humans. Affected RyRs are more active than normal, leaking Ca from the SR during diastole causing arrhythmias and dysfunction. FKBP12.6 and calmodulin (CaM) are proteins that bind tightly with and may stabilize RyR2. We have developed novel quantitative methods to assess (in adult cardiac myocytes) how FKBP, CaM and other peptides bind to and modulate RyR2 gating, and are structurally positioned on the myocyte RyR2. While FKBP12.6 binds RyR2 with high affinity, it is not a critical RyR2 regulator. Here we examine how CaM binding and altered domain-domain interaction in RyR2 are involved in disease-related RyR2 dysfunction, using fluorescent tagged proteins and novel targeted sensors in adult ventricular myocyte confocal imaging to address 4 aims. We will: 1) Test whether HF-related RyR alterations decrease CaM binding and increase the access of the structural unzipping peptide DPc10. 2) Test whether known RyR inhibitors work on pathological RyR2 by altering FKBP12.6, CaM or DPc10 binding. 3) Measure Cleft [Ca] using novel targeted Ca sensors. 4) Enhance RyR2 structural model by mapping sites of S100A1, CaMKII, IPTx and Sorcin. This is a highly collaborative project between two groups with shared interests in cardiac calcium regulation in HF and arrhythmias and with complementary technical expertise in physiology, pharmacology, biochemistry, molecular biology, fluorescence spectroscopy and confocal imaging. This will greatly enhance our understanding of RyR2 structure and function in cardiac myocytes in health and disease and provide novel strategies for the development of therapeutics for pathologically modulated RyR2 in the heart.